PROJECT SUMMARY Bladder cancer is a common cancer of the urinary tract with 79,000 new cases diagnosed in the US each year. A bladder tumor may be low-grade or high-grade. While low-grade tumors are most often confined to the urothelial layer, high-grade tumors often invade into the muscle layer of the bladder. A tumor that is confined to the urothelial layer is resected from the bladder; however, muscle invasive tumors require surgical removal of the bladder and adjuvant chemotherapy if there is metastasis. This high metastatic potential of high-grade tumors is responsible for the significant morbidity and mortality associated with bladder cancer. Two-thirds of patients with high-grade tumors have muscle-invasive disease at initial presentation. Fifty percent of patients with muscle- invasive bladder cancer will develop metastasis within two years, and the five-year survival of patients with metastatic disease is only 15%, despite chemotherapy. Identification of tumor promoting molecular pathways in bladder cancer could lead to the development of molecular markers that can identify patients who are likely to develop metastasis, and individualize selection of chemotherapy regimens. This could significantly improve the clinical management of patients with muscle-invasive disease. Proteoglycans have been shown to regulate tumor growth, progression, and chemoresistance. However, the role of proteoglycan-degrading enzymes (PDEs) is not well understood in benign or malignant diseases. Identification and characterization of a novel PDE showed that expression of this enzyme promotes malignant phenotype and chemotherapeutic resistance in bladder cancer and normal urothelial cells. The expression of this PDE was elevated in bladder tumor tissues and correlated with malignant progression of the disease and response to adjuvant chemotherapy. This project is designed to test a hypothesis that the activity of the PDE and its downstream effectors drive a malignant phenotype (muscle invasion, metastasis) and chemoresistance in bladder cancer and associate with poor clinical outcome. The hypothesis will be tested by mapping the functional domains in the PDE that are responsible for its enzymatic and biological activities and identifying downstream effectors that drive the PDE-induced malignant behavior (Aim 1). The expression and the activity of the PDE together with molecular effectors identified in Aim 1 will be validated in bladder cancer specimens and correlated with clinical outcome (Aim 2). Impact: This project will be the first study to evaluate a novel PDE that is potentially a molecular determinant of advanced bladder cancer and chemoresistance. Understanding the mechanism by which PDE induces malignant phenotype and chemoresistance, and the association of the PDE and its effectors with clinical outcome, if successful, may improve clinical management patients through the development of new molecular markers and therapeutic targets for aggressive bladder cancer.